PKC-dependent Regulation of NAG-1/PLAB/MIC-1 Expression in LNCaP Prostate Carcinoma Cells

Minsub Shim and Thomas E. Eling1 Eicosanoids Biochemistry Section, Laboratory of Molecular Carcinogenesis, NIEHS,

National Institutes of Health, RTP NC 27709 Running Title; PKC, NAG-1, and apoptosis

1 To whom correspondence should be addressed: Thomas E. Eling, Laboratory of Molecular

Carcinogenesis, NIEHS, NIH, 111 TW Alexander Drive, Research Triangle Park, NC 27709,

Phone : (919) 541-3911, Fax : (919) 541-0146, email : eling@niehs.nih.gov

NAG-1 (nonsteroidal anti-inflammatory drug-activated gene), a member of TGF beta superfamily, is involved in cellular processes such as inflammation, apoptosis/survival, and tumorigenesis and is regulated by p53, Sp1, and Egr-1. In the current study, the regulation of NAG-1 expression in LNCaP human prostate carcinoma cells by TPA (12-O-tetradecanoylphorbol-13-acetate) was examined. TPA treatment increased NAG-1 protein and mRNA levels in a time and concentration-dependent manner as well as

NF-κB binding/transcriptional activity in LNCaP cells. Pre-treatment with PKC (protein kinase C) inhibitor blocked TPA-induced increase in NAG-1 protein levels and

NF-κB binding/transcriptional activity while an inhibition of p38, JNK, or MEK activity had no effect on TPA-induced NAG-1 levels

and NF-κB transcriptional activity. Expression of constitutively active PKCs

induced an increase in NF-κB transcriptional activity and NAG-1 protein levels in LNCaP

cells. The expression of NF-κB p65 induced NAG-1 promoter activity and chromatin immunoprecipitaion assay for p65 showed

that NF-κB binds NAG-1 promoter in LNCaP cells. Inhibition of TPA-induced NAG-1 expression by NAG-1 Si RNA blocked TPA-induced apoptosis in LNCaP cells, suggesting induction of NAG-1 negatively affects LNCaP cell survival. These results demonstrate that NAG-1 expression is up-regulated by TPA in LNCaP cells through a PKC-dependent

pathway involving the activation of NF-κB.

INTRODUCTION

Apoptosis, the programmed cell death, is critical for cellular homeostasis and prevention of tumor development. In the prostate carcinoma, cells are initially dependent upon androgens for growth and survival while they become resistant to apoptotic stimuli such as androgen deprivation in the advanced stages of prostate carcinoma (1). LNCaP (2), an androgen responsive human prostate carcinoma cell line, has been used as a model for progression of prostate carcinoma from androgen sensitive to insensitive stage. TPA (12-O-tetradecanoylphorbol-13-acetate), an agonist of protein kinase C (PKC), induces

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apoptosis in LNCaP (3-5). Several mechanisms have been suggested for this TPA-induced apoptosis in LNCaP cells. Powell et al (4) showed persistent membrane translocation of

PKCα during TPA-induced apoptosis of LNCaP cells while Fuji et al (6) demonstrated that PKCδ mediates apoptotic responses to TPA in LNCaP cells. However, the functional downstream effectors of PKC in TPA-induced apoptosis of LNCaP cells have not been elucidated. In addition, the involvement of MAPK kinase pathways, such as JNK (7) or p38 (8) as well as Akt pathways (8) also have been proposed for TPA-induced apoptosis of LNCaP cells.

PKC is a family of serine/threonine kinases and regulates a variety of cellular processes including mitogenesis, differentiation, apoptosis, and tumor promotion (9-13). PKC family is comprised of isoforms classified into three groups based on their structure and

regulation. The conventional PKCs (α, β1, β2, γ) require calcium and are DAG (or TPA)-responsive. The novel PKCs (δ, ε, η, θ) are calcium-independent but activated by DAG (or

TPA). The atypical PKCs (λ, ζ) are both calcium and DAG (or TPA)-independent. PKCs regulate cellular responses by inducing the expression of enzymes such as nitric oxide synthase or cyclooxygenase-2 as well as the activation of transcription factors such as activator protein-1 (AP-1) or nuclear factor-

kappaB (NF-κB). NF-κB is a transcription factor which

mediates the immune responses, inflammation, proliferation, and apoptosis by regulating the expression of cytokines, growth factors, and

adhesion molecules. NF-κB functions as homo- or heterodimers among Rel family proteins, all of which shares Rel homology domains. There are five members of Rel family identified: p50 (p105), p52 (p100), Rel A (p65), c-Rel, and Rel B. Inducible form of NF-

κB is a heterodimer between p50 and p65 and NF-κB can be activated by a variety of stimuli through a cascade of kinase signaling, which

leads to release of NF-κB from IκB, an inhibitory protein, and subsequent translocation to nucleus. Many studies have shown that

apoptotic signal activates NF-κB and activation of NF-κB protects cells from apoptosis. However, other data indicate NF-κB promotes apoptosis. For example, NF-κB activates Fas ligand (FasL), a member of tumor necrosis factor family, promoter during activation- or chemotherapeutic agent-induced apoptosis in T

lymphocytes (14,15). Inhibition of NF-κB in LNCaP cells renders cells resistant to apoptosis induced by death ligands in the presence or absence of irradiation (16). Recently, Fujioka

et al have shown that NF-κB is essential in activating p53 to initiate proapototic signaling in reponse to overgeneration of superoxide (17). Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a distant member of

TGF β superfamily (18) and is also known as macrophage inhibitory cytokine-1 (MIC-1) (19), placental transforming growth factor beta

(PTGF-β) (20), prostate derived factor (PDF) (21), novel placental bone morphogenic protein (PLAB) (22), or growth differentiation factor-15 (GDF-15) (23). NAG-1 was identified from a cyclooxygenase inhibitor (indomethacin)-treated

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HCT-116 colon cancer cell lines using subtractive hybridization (18). NAG-1 is highly expressed in human placenta and prostate while it is expressed in kidney and pancreas at a lower level (21). NAG-1 protein is synthesized as a 308 amino acid propeptide with RXXR cleavage site and secreted as a 30 kD dimeric mature protein after cleavage at RXXR site and formation of disulfide bond between cysteine residues. Pro-region of NAG-1 has been shown to be N-glycosylated during the processing of the NAG-1 through ER and Golgi (24). NAG-1 regulates a wide range of cellular functions in distinct cellular contexts. For example, NAG-1 is induced by TPA treatment in macrophages and blocks the late stage of macrophage activation (19). NAG-1 inhibits proliferation of breast carcinoma cells (25), mink lung epithelial cells, and prostate carcinoma cells (26). Treatment with NAG-1 induced a decrease in cell adhesion and increase in apoptosis in prostate cancer cell line (27) and forced expression of NAG-1 in HCT-116 colon cancer cells resulted in reduced soft agar growth and tumor growth in nude mice (18). In addition, NAG-1 has been identified as a p53 target gene (25,26). In contrast to the role of NAG-1 as an anti-tumorigenic, pro-apoptotic molecule, several reports have shown that NAG-1 is positively associated with tumor development. Up-regulation of NAG-1 has been reported in serum from patients with metastatic breast, prostate, and colorectal carcinomas (28) and in transition from androgen-dependent to androgen independent prostate carcinoma cells (29).

In this study, we examined the regulation of NAG-1 expression during TPA-induced apoptosis in LNCaP human prostate carcinoma cells. TPA induced NAG-1

expression as well as NF-κB activation in LNCaP cells by a PKC dependent mechanism. We found that p65 directly binds/activates NAG-1 promoter and inhibition of NAG-1 expression diminished TPA-induced apoptosis in LNCaP cells. These data suggest that the expression of NAG-1 provides the novel mechanism for understanding the downstream effectors for TPA-induced apoptosis in LNCaP cells.

MATERIALS AND METHODS

Materials Akt antibody and phospho-Akt antibody were purchased from Cell Signaling Technology. Ly294002, GF109203X, PD98059, SB203580, and SP600125 were purchased from Calbiochem. TPA was purchased from Sigma.

Cell culture LNCaP human prostate carcinoma cells were purchased from American Type Culture Collection and cultured in RPMI1640 media supplemented with 10 % fetal bovine serum

(Hyclone) and 10 µg/ml gentamycin (Invitrogen). Western blot analysis Cells were lysed in RIPA buffer and protein concentration was measured by BCA reagent (Pierce). Equal amounts of protein

were solubilized and heated at 65°C in LDS sample buffer (Invitrogen) with sample reducing agent (Invitrogen) for 10 min, and then separated by SDS-PAGE. The separated proteins were transferred to an Immobilon-P membrane

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(Millipore). Following incubation in blocking buffer (TBS with 5 % non fat dry milk and 0.1 % Tween-20) for 1 hr at room temperature,

the membranes were probed for overnight at 4°C with rabbit polyclonal anti-NAG-1 (1:2000), monoclonal anti-actin (Sigma, 1:5000), or anti-HA (Roche, 1:2000) antibody. The membranes were washed and then probed with an HRP-linked secondary antibody (Amersham, 1:2500) for 1 hr at room temperature. Detection was made with an enhanced chemiluminescence reagent followed by exposure of membrane to film.

Northern blot analysis Total RNA was isolated using Promega’s SV total RNA isolation kit or Qiagen’s mRNA isolation kit. NAG-1 cDNA

was labeled with [α-32P] dCTP by using Decaprime II kit (Ambion) according to manufacturer’s protocol. RNA was electrophoresed on agarose gel containing formaldehyde, transferred to Hybond XL membrane (Amersham) and UV cross-linked.

Blots were incubated at 65°C in Rapid-hybridization buffer (Amersham) and sequentially washed with washing buffer 1 (0.5 % SDS, 0.1x SSC) and washing buffer 2

(0.1 % SDS, 0.1x SSC) at 65°C. Films were exposed to membranes at –80°C and developed. Transfection and luciferase assay

pcDNA3.1-HA-PKCα ∆NPS and pcDNA3.1-HA-PKCδ ∆NPS were a kind gift from Dr. Jae-Won Soh (Inha University, Incheon,

Korea). NF-κB p65 expression plasmid was a kind gift from Dr. Dean Ballard (Vanderbilt University School of Medicine, Nashville,

Tennessee). Construction of NAG-1 reporter plasmid has been described previously (30).

LNCaP cells were plated at 1.25 x 105 cells/well in 12 well culture plate. Two days after plating, LNCaP cells were transfecetd in triplicate with the specified reporter plasmid as described in the text and pRL-null plasmid using Lipofectamine plus (Invitrogen) according to manufacturer’s protocol. Forty-eight hours later, cells were harvested and the luciferase activity was determined and normalized to Renilla luciferase activity with a dual luciferase assay kit (Promega).

Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared as previously described by Schreiber et al (31). Nuclear extracts were incubated at room

temperature for 30 minutes with 10 µl of master

binding mix with 32P-labeled NF-κB probe (Santa Cruz). Samples were loaded onto 6% DNA retardation gel (Invitrogen) and subjected to electrophoresis in 0.25X TBE buffer at 100V for 1 hour. The gel was transferred to

Whatman paper, dried in a 80oC gel dryer for 1 hour, then exposed to film for appropriate time to check the DNA binding pattern.

Generation of PKC isoform expressing LNCaP cells

φNX cells were cultured with DMEM medium plus 10% FBS and transfected with pRev-Tet-On (BD Clontech) by Lipofectamine2000 (Invitrogen) according to manufacturer’s protocol. Virus containing

medium was collected from φNX cell cultures 3 times, 6 hours apart, filtered through a 0.45 µm

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filter, and overlaid onto LNCaP cells with 10

µg/ml polybrene (Sigma). Cultures were maintained with viral containing medium overnight and 24 hrs later cells were shifted to a

selection medium containing 500 µg/ml G418. A pool of G418-resistant LNCaP cells (Tet-On LNCaP) was infected with virus containing

medium from φNX cells which were transfected with pRev-TRE-PKCα ∆NPS or pRev-TRE-PKCδ ∆NPS. Cells were subjected to a selection with 500 µg/ml hygromycin and 250 µg/ml G418. Hygromycin/G418 resistant cells were pooled and used for western blot analysis.

Chromatin immunoprecipitation (CHIP) assay LNCaP cells were plated on 60 mm culture dishes at 1 x 10 6 cells / dish and 24 hour later, were fixed by the addition of 1% formaldehyde in PBS for 10 min. The cells were scraped and collected by centrifugation. Cells were lysed in lysis buffer and p65-associated chromatins were immunoprecipitated using CHIP assay kit (Upstate) according to manufacturer’s protocol. After phenol/chlorform extraction and ethanol

precipitation, the pellet was resuspended in 50 µl of H2O. For the PCR reaction, 5 µl of immunoprecipitated DNA or total input DNA was amplified using primers for NAG-1 promoter sequence.

Generation of NAG-1 Si RNA expressing LNCaP cells The NAG-1 Si RNA vector (pSuper-retro-puro-Si NAG1) was constructed using a pSuper-retro-puro and a synthetic olgonucleotide targeting 5’-ACATGCACGCGCAGATCAA-3’ corresponding to positions from 780 to 798 on

NAG-1 mRNA. φNX cells were cultured with DMEM medium plus 10% FBS and transfected with pSuper-retro-puro-Si NAG1 by Lipofectamine2000 (Invitrogen) according to manufacturer’s protocol. Virus containing

medium was collected from φNX cell cultures 3 times, 6 hours apart, filtered through a 0.45 µm filter, and overlaid onto LNCaP cells with 10

µg/ml polybrene (Sigma). Cultures were maintained with viral containing medium overnight and 24 hrs later cells were shifted to a

selection medium containing 2 µg/ml puromycin (Sigma). A pool of puromycin-resistant LNCaP cells were pooled and used for western blot analysis.

RESULTS

TPA increases NAG-1 mRNA and protein levels in LNCaP cells

To study the role of NAG-1 during TPA-induced apoptosis in LNCaP cells, we first determined the effect of TPA on NAG-1 levels in LNCaP cells. LNCaP cells were treated with various concentrations of TPA and twenty-four hour after TPA treatment, total cell lysates were prepared and NAG-1 protein levels were determined by western blot analysis. As shown in Fig. 1A, TPA induced an increase in NAG-1 protein levels in a concentration-dependent manner. TPA also induced an increase in NAG-1 protein in a time-dependent manner, reaching the maximum level at 12 hour after TPA treatment (Fig. 1B). To examine if this increase in NAG-1 protein levels results from increased NAG-1 mRNA levels, we performed

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northern blot analysis for NAG-1. TPA treatment caused an increase in NAG-1 mRNA levels in a concentration- and time-dependent manner (Fig. 1C, D), indicating that TPA regulates NAG-1 levels at the transcriptional level. To ascertain if TPA treatment activates NAG-1 promoter, we transfecetd LNCaP cells with a luciferase reporter gene fused to NAG-1 promoter (-966/+70) and then treated these cells with TPA. TPA treatment resulted in an increase in NAG-1 promoter activity in a concentration-dependent manner over that observed in cells treated with vehicle (Fig. 1E). These results demonstrate that TPA regulates NAG-1 protein levels at the transcriptional level.

TPA induces NAG-1 protein by a PKC-dependent mechanism in LNCaP cells While TPA has been shown to activate PKCs in LNCaP cell (6,32), TPA can affect various signaling pathways via the activation/inhibition of other kinases, such as JNK (7), p38 (8), and Akt (8) in LNCaP cells. To test if TPA induces NAG-1 expression by a PKC-dependent mechanism, LNCaP cells were pre-treated with GF109230X which is reported to be an inhibitor of PKC (33). LNCaP cells were then treated with TPA and total lysates were subjected to western blot analysis. While GF109230X pre-treatment completely blocked TPA-induced NAG-1 protein expression, pre-treatment with JNK inhibitor (SP600125), p38 kinase inhibitor (SB203580), or MEK inhibitor (PD98059) did not affect TPA-induced NAG-1 expression (Fig. 2A). Tanaka et al (8) showed that TPA induces apoptosis in LNCaP cells by

activation of p38 MAPK and inhibition of Akt. In addition, Yamaguchi et al (34) recently showed that NAG-1 is a downstream target of Akt/GSK3 pathway in HCT-116 human

colorectal carcinoma cells. In order to examine if TPA-induced increase in NAG-1 protein levels results from an inhibition of Akt, we treated LNCaP cells with phosphatidyl-3 kinase (PI3K) inhibitor, Ly294002 (35) and total cell lysates were subjected to western blot analysis. Since PI3K is an immediate upstream kinase of Akt, Ly294002 treatment blocks the phosphorylation of Akt. While TPA or Ly294002 treatment blocked the phosphorylation of Akt, Ly294002 treatment did not affect NAG-1 protein levels, suggesting an inhibition of Akt is not involved in TPA-induced increase in NAG-1 protein levels (Fig 2B) in LNCaP cells. These results indicate that the induction of NAG-1 by TPA is dependent on PKCs.

TPA induces NF-κB activity in LNCaP cells We have cloned the promoter region of NAG-1 and subsequent sequence analysis

identified potential NF-κB binding sites in NAG-1 promoter. TPA activates NF-κB (36,37) and Rameshwar et al have shown that

NF-κB positively regulates TGF-β1 expression from monocytes with idiopathic myelofibrosis

(38). In order to identify if NF-κB is associated with TPA-induced NAG-1 expression, LNCaP cells were treated with TPA and nuclear extracts were isolated at various time points and subjected to EMSA. As shown in Fig. 3A, TPA

treatment significantly increased NF-κB binding

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activity. To ascertain if the TPA-induced

increase in NF-κB binding activity was also accompanied by an increase in NF-κB transactivation activity, we transfected LNCaP

cells with pκB-luciferase, a reporter plasmid which has three consecutive NF-κB binding sites upstream of luciferase, and then treated these cells with TPA. As shown in Fig. 3B, TPA treatment resulted in a significant increase in

NF-κB transcriptional activity. In order to determine if PKCs are associated with TPA-

induced increase in NF-κB transcriptional activity, we transfected LNCaP cells with pκB-luciferase reporter plasmid and treated with GF109230X in the presence or absence of TPA. While GF109230X treatment blocked TPA-

induced increase in NF-κB transcriptional activity, JNK inhibitor, p38 kinase inhibitor, or MEK inhibitor did not affect TPA-induced NF-

κB activation (Fig 3B). To provide further evidence that PKCs activate NF-κB transcriptional activity in LNCaP cells, we determined the effect of PKC expression on NF-

κB transcriptional activity. Powell et al (4) showed that four PKC isoforms (-α, -δ, -ε, -η) are expressed in LNCaP cells among classical

and novel PKC isoforms. Since PKC α and δ have been shown to be involved in TPA-induced apoptosis in LNCaP cells, we co-transfected

LNCaP cells with NF-κB reporter plasmid and constitutively active form of PKC α or δ (39). As shown in Fig. 3C, constitutively active PKC

α and δ activated NF-κB reporter, while expression of constitutively active PKC δ caused more significant increase in NF-κB transcriptional activity. These results

demonstrate that TPA-induced activation of NF-

κB is dependent on PKCs in LNCaP cells.

NF-κB activates NAG-1 promoter Since TPA induced an increase in

NAG-1 levels as well as NF-κB transcriptional activity in LNCaP cells, we examined the direct

effect of NF-κB on NAG-1 promoter. LNCaP cells were co-transfected with p65 expression plasmid and NAG-1 promoter reporter construct. Expression of p65 induced 4-fold increase in NAG-1 promoter activity while Egr-1, which has been shown to mediate troglitazone-induced increase in NAG-1 level (40), increased NAG-1 promoter activity 2-fold (Fig. 4A). In order to

localize NF-κB binding site(s) in NAG-1 promoter, we co-transfected LNCaP cells with different length of NAG-1 promoter reporter plasmid and p65 expression plasmid. NAG-1 promoter activation by p65 was most significant in reporter plasmid which has –474/40 region of NAG-1 promoter, indicating that this region of

NAG-1 promoter (-133 to –474) has NF-κB binding site(s) (Fig. 4B). NF-κB binding to NAG-1 promoter in vivo (in LNCaP cells) was determined by chromatin immunoprecipitation (CHIP) assay. LNCaP cells were collected after formaldehyde treatment and isolated chromatin was subjected to sonication followed by an immunoprecipitation with antibodies

against NF-κB p65 subunit. After immunoprecipitation, NAG-1 promoter region from -309 to +63 was amplified (Fig. 4C),

indicating that NF-κB binds to NAG-1 promoter in LNCaP cells. These results demonstrate that

NF-κB binds and activates NAG-1 promoter.

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PKC induces NAG-1 in LNCaP cells To examine the direct effect of PKCs on NAG-1 levels, we measured NAG-1 levels in LNCaP cells which express constitutively active

PKC α or δ isoform. In order to make the pools of LNCaP cells that express constitutively active PKC, we used retrovirus-mediated, tetracycline-inducible (tet-on) system. LNCaP cells were infected with reverse TetR encoding retroviruses and selected with G418, followed by another retroviral infection for HA-tagged each PKC isoform and selection with hygromycin. Cell lysates were made and subjected to western blot analysis for NAG-1, followed by western blot analysis for HA and actin. The pooled cells expressed high level of constitutively active PKC without the addition of tertracycline and this may be due to the use of pooled cells rather than a single clone. As shown in Fig. 5, forced

expression of constitutively active PKC α or δ resulted in induction of NAG-1, while

constitutively active PKC δ was more potent in inducing NAG-1 protein levels, indicating PKC is the upstream kinase for NAG-1 expression in LNCaP cells.

Inhibition of NAG-1expression blocks TPA-induced apoptosis in LNCaP cells In order to examine the biological significance of NAG-1 induction by TPA in LNCaP cells, we generated LNCaP cells which express NAG-1 Si RNA using retroviral system. To determine if the expression of NAG-1 Si RNA can block TPA-induced NAG-1 expression, vector or NAG-1 Si RNA infected LNCaP cells

were pooled after the selection with puromycin and treated with different concentrations of TPA. Twenty-four hour after TPA treatment, total lysates were made and subjected to western blot analysis for NAG-1. As shown in Fig. 6A, the stable expression of NAG-1 Si RNA partially blocked TPA-induced NAG-1 expression in the pool of LNCaP cells infected with NAG-1 Si RNA containing retrovirus. To determine the effects of NAG-1 induction by TPA on LNCaP cell survival, vector or NAG-1 Si RNA containing retrovirus infected LNCaP cells were plated and treated with various concentrations of TPA for 24 hours. In vector-infected LNCaP cells, 2.5 ng/ml TPA treatment resulted in ~ 50 % reduction in cell number compared to control cells, while stable expression of NAG-1 Si RNA resulted in 20 % reduction in cell number after 2.5 ng/ml TPA treatment (Fig 6B), suggesting the inhibition of NAG-1 expression renders LNCaP cells resistant to TPA-induced apoptosis. In addition, the stable expression of NAG-1 Si RNA increased the rate of basal proliferation which is consistent with the role of NAG-1 as an inducer of growth arrest/apoptosis. In order to provide additional evidence for the role of NAG-1 in TPA-induced apoptosis in LNCaP cells, vector or NAG-1 Si RNA infected pool of LNCaP cells were treated with various concentrations of TPA for 24 hours and DNA content was measured by FACS analysis. In all concentrations of TPA tested, inhibition of TPA-induced NAG-1 expression by NAG-1 Si RNA resulted in less amount (30 ~ 40 %) of sub-G1 phase compared to vector infected cells (data not shown). Together, these results suggest that the

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induction of NAG-1 may be one of the mechanisms by which TPA induces apoptosis in LNCaP cells.

DISCUSSION

NAG-1 is a divergent member of TGF-

β superfamily and has been shown to be induced by NSAIDs in our laboratory. The major role of NAG-1 is not clear, however, several studies suggest NAG-1 as an inducer of growth arrest or apoptosis in addition to other cellular functions. In this report, we showed that TPA induces NAG-1 protein by a PKC-dependent mechanism

involving the activation of NF-κB and this TPA-induced NAG-1 expression in LNCaP cells negatively affects cell survival.

TPA induced the expression of NAG-1 protein and mRNA in a concentration- and time-dependent manner. TPA-induced NAG-1 expression was significantly blocked by pre-treatment with PKC-inhibitor, indicating that TPA induces NAG-1 expression by a PKC-dependent mechanism. In addition, forced

expression of constitutively active PKCα or PKCδ in LNCaP cells induced NAG-1 expression, demonstrating PKC is an upstream signaling kinase in TPA-induced NAG-1

expression. Since PKCα and PKCδ are involved in TPA-induced apoptosis in LNCaP cells, the fact that the expression of

constitutively active PKCα or PKCδ induces NAG-1 suggests that NAG-1 may be the downstream target in TPA-induced apoptosis in LNCaP cells. The expression of constitutively

active PKCα or PKCδ in LNCaP cells induced

growth inhibition as well as NAG-1 expression compared to parental cell line (pTet-On LNCaP), suggesting NAG-1 expression may be involved in growth inhibition in LNCaP cells that express

constitutively active PKCα or PKCδ (data not shown).

TPA treatment activated NF-κB binding and transcription activity by a PKC dependent mechanism and the transient expression of

constitutively active PKCs activated NF-κB activity in LNCaP cells. We also have shown that p65 binds and activates NAG-1 promoter using CHIP assay and NAG-1 promoter reporter assay. TPA treatment induced an increase in binding activity of other transcription factors that have putative binding site(s) on NAG-1 promoter such as AP-1, Egr-1, and C/EBP (data not shown). While other transcription factors can be activated by TPA, co-transfection with NAG-1 promoter reporter showed that p65 was most efficient at activating NAG-1 promoter (Fig. 4A and data not shown). However, co-

transfection of LNCaP cells with Iκ-Bα and NAG-1 promoter reporter resulted in a partial inhibition of TPA-induced increase in NAG-1 promoter activity (data not shown), suggesting other transcription factors are also involved in TPA-induced NAG-1 expression. NAG-1 promoter has two p53 binding sites and NAG-1 is induced by signals that activate p53 (25,26). While LNCaP cell has wild type p53, PC-3, an androgen-insensitive prostate carcinoma cell, has truncated p53 (p53-null). In order to determine if TPA-induced NAG-1 expression is associated with the activation of p53, we treated PC-3 cells with same concentrations of TPA as in LNCaP

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and performed western blot analysis for NAG-1. TPA treatment also induced NAG-1 protein in PC-3 cells (data not shown), indicating TPA-induced NAG-1 expression is p53-independent.

When we performed western blot for NAG-1 in LNCaP cells, we found that unprocessed NAG-1 protein (38 kD) migrates as a doublet on SDS-PAGE (Fig 1A, B). We also observed the existence of NAG-1 protein doublet in lysates from other cell lines, such as HCT-116 colon cancer cell line. NAG-1 protein is N-glycosylated at Asn 41 and has the potential phosphorylation sites. Therefore, it is possible that this difference in electrophoretic mobility of unprocessed NAG-1 protein may result from the differential post-translational modifications. However, the treatment of LNCaP lysates with glycosidases or phosphatases did not affect the migration of unprocessed NAG-1 protein as a doublet (data not shown) on SDS-PAGE, suggesting post-translational modifications are not involved in differential electrophoretic mobility of NAG-1 protein. It is also possible that translation of NAG-1 protein may start at several sites leading the generation of polypeptide with different length since NAG-1 mRNA has several in-frame ATG codons thus providing alternative translational start sites. Further study will determine the biological significance of NAG-1 doublet and the mechanism by which NAG-1 doublet is generated.

NAG-1 has been shown to negatively affect cell growth or survival in epithelial or other cancer cell lines and believed to mediate

the effect of several chemopreventive compounds (18,27,30,40). We have shown that the pool of LNCaP cells that stably express NAG-1 Si RNA was more resistant to the apoptotic effect of TPA, indicating that NAG-1 is associated with TPA-induced apoptosis in LNCaP cells. Along with TPA, a thapsigargin treatment has been known to induce apoptosis in LNCaP cells (7). When LNCaP cells were incubated with thapsigargin for 48 hours, massive apoptosis was observed but no NAG-1 expression was induced (data not shown), suggesting the increased expression of NAG-1 is not a consequence of apoptosis. In contrast to inducing apoptosis in LNCaP cells, TPA treatment induced an increase in cell growth as well as a decrease in NAG-1 levels in HCT-116 colon cancer cell lines (data not shown), supporting the notion that NAG-1 negatively affects cell growth/survival. In addition, PC-3 clones that stably transfected with NAG-1 displayed impaired growth and increased apoptosis (unpublished data).

In conclusion our results demonstrate that TPA induces NAG-1 in LNCaP cells via a PKC-dependent pathway involving the

activation of NF-κB. In addition, our finding identifies NAG-1 as a downstream molecule in TPA-induced apoptosis and contributes to the understanding of apoptotic pathways which can provide the approaches to prostate cancer therapeutics.

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REFERENCES

1. Feldman, B. J., and Feldman, D. (2001) Nat Rev Cancer 1, 34-45 2. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P., Rosenthal, H., Chu, T. M., Mirand,

E. A., and Murphy, G. P. (1983) Cancer Res 43, 1809-1818 3. Garzotto, M., White-Jones, M., Jiang, Y., Ehleiter, D., Liao, W. C., Haimovitz-Friedman, A.,

Fuks, Z., and Kolesnick, R. (1998) Cancer Res 58, 2260-2264 4. Powell, C. T., Brittis, N. J., Stec, D., Hug, H., Heston, W. D., and Fair, W. R. (1996) Cell

Growth Differ 7, 419-428 5. Zhao, X., Gschwend, J. E., Powell, C. T., Foster, R. G., Day, K. C., and Day, M. L. (1997) J

Biol Chem 272, 22751-22757 6. Fujii, T., Garcia-Bermejo, M. L., Bernabo, J. L., Caamano, J., Ohba, M., Kuroki, T., Li, L.,

Yuspa, S. H., and Kazanietz, M. G. (2000) J Biol Chem 275, 7574-7582 7. Engedal, N., Korkmaz, C. G., and Saatcioglu, F. (2002) Oncogene 21, 1017-1027 8. Tanaka, Y., Gavrielides, M. V., Mitsuuchi, Y., Fujii, T., and Kazanietz, M. G. (2003) J Biol

Chem 278, 33753-33762 9. Liu, W. S., and Heckman, C. A. (1998) Cell Signal 10, 529-542 10. Blobe, G. C., Obeid, L. M., and Hannun, Y. A. (1994) Cancer Metastasis Rev 13, 411-431 11. Spitaler, M., and Cantrell, D. A. (2004) Nat Immunol 5, 785-790 12. Parker, P. J., and Murray-Rust, J. (2004) J Cell Sci 117, 131-132 13. Dempsey, E. C., Newton, A. C., Mochly-Rosen, D., Fields, A. P., Reyland, M. E., Insel, P. A.,

and Messing, R. O. (2000) Am J Physiol Lung Cell Mol Physiol 279, L429-438 14. Kasibhatla, S., Genestier, L., and Green, D. R. (1999) J Biol Chem 274, 987-992 15. Kasibhatla, S., Brunner, T., Genestier, L., Echeverri, F., Mahboubi, A., and Green, D. R.

(1998) Mol Cell 1, 543-551 16. Kimura, K., and Gelmann, E. P. (2002) Cell Death Differ 9, 972-980 17. Fujioka, S., Schmidt, C., Sclabas, G. M., Li, Z., Pelicano, H., Peng, B., Yao, A., Niu, J., Zhang,

W., Evans, D. B., Abbruzzese, J. L., Huang, P., and Chiao, P. J. (2004) J Biol Chem 279, 27549-27559

18. Baek, S. J., Kim, K. S., Nixon, J. B., Wilson, L. C., and Eling, T. E. (2001) Mol Pharmacol 59, 901-908

19. Bootcov, M. R., Bauskin, A. R., Valenzuela, S. M., Moore, A. G., Bansal, M., He, X. Y., Zhang, H. P., Donnellan, M., Mahler, S., Pryor, K., Walsh, B. J., Nicholson, R. C., Fairlie, W.

D., Por, S. B., Robbins, J. M., and Breit, S. N. (1997) Proc Natl Acad Sci U S A 94, 11514-11519

11

by guest on Novem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

20. Lawton, L. N., Bonaldo, M. F., Jelenc, P. C., Qiu, L., Baumes, S. A., Marcelino, R. A., de Jesus, G. M., Wellington, S., Knowles, J. A., Warburton, D., Brown, S., and Soares, M. B.

(1997) Gene 203, 17-26 21. Paralkar, V. M., Vail, A. L., Grasser, W. A., Brown, T. A., Xu, H., Vukicevic, S., Ke, H. Z., Qi,

H., Owen, T. A., and Thompson, D. D. (1998) J Biol Chem 273, 13760-13767 22. Hromas, R., Hufford, M., Sutton, J., Xu, D., Li, Y., and Lu, L. (1997) Biochim Biophys Acta

1354, 40-44 23. Strelau, J., Bottner, M., Lingor, P., Suter-Crazzolara, C., Galter, D., Jaszai, J., Sullivan, A.,

Schober, A., Krieglstein, K., and Unsicker, K. (2000) J Neural Transm Suppl, 273-276 24. Bauskin, A. R., Zhang, H. P., Fairlie, W. D., He, X. Y., Russell, P. K., Moore, A. G., Brown, D.

A., Stanley, K. K., and Breit, S. N. (2000) Embo J 19, 2212-2220 25. Li, P. X., Wong, J., Ayed, A., Ngo, D., Brade, A. M., Arrowsmith, C., Austin, R. C., and

Klamut, H. J. (2000) J Biol Chem 275, 20127-20135 26. Tan, M., Wang, Y., Guan, K., and Sun, Y. (2000) Proc Natl Acad Sci U S A 97, 109-114 27. Liu, T., Bauskin, A. R., Zaunders, J., Brown, D. A., Pankhurst, S., Russell, P. J., Breit, S. N.,

and Panhurst, S. (2003) Cancer Res 63, 5034-5040 28. Welsh, J. B., Sapinoso, L. M., Kern, S. G., Brown, D. A., Liu, T., Bauskin, A. R., Ward, R. L.,

Hawkins, N. J., Quinn, D. I., Russell, P. J., Sutherland, R. L., Breit, S. N., Moskaluk, C. A.,

Frierson, H. F., Jr., and Hampton, G. M. (2003) Proc Natl Acad Sci U S A 100, 3410-3415 29. Karan, D., Kelly, D. L., Rizzino, A., Lin, M. F., and Batra, S. K. (2002) Carcinogenesis 23,

967-975

30. Baek, S. J., Wilson, L. C., and Eling, T. E. (2002) Carcinogenesis 23, 425-434 31. Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic Acids Res 17,

6419 32. Garcia-Bermejo, M. L., Leskow, F. C., Fujii, T., Wang, Q., Blumberg, P. M., Ohba, M.,

Kuroki, T., Han, K. C., Lee, J., Marquez, V. E., and Kazanietz, M. G. (2002) J Biol Chem 277, 645-655

33. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V.,

Boissin, P., Boursier, E., Loriolle, F., and al., e. (1991) J Biol Chem 266, 15771-15781 34. Yamaguchi, K., Lee, S. H., Eling, T. E., and Baek, S. J. (2004) J Biol Chem

35. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994) J Biol Chem 269, 5241-5248 36. Sun, S. C., Ganchi, P. A., Ballard, D. W., and Greene, W. C. (1993) Science 259, 1912-1915 37. Henkel, T., Machleidt, T., Alkalay, I., Kronke, M., Ben-Neriah, Y., and Baeuerle, P. A. (1993)

Nature 365, 182-185 38. Rameshwar, P., Narayanan, R., Qian, J., Denny, T. N., Colon, C., and Gascon, P. (2000) J

Immunol 165, 2271-2277

12

by guest on Novem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

39. Soh, J. W., and Weinstein, I. B. (2003) J Biol Chem 278, 34709-34716 40. Baek, S. J., Kim, J. S., Nixon, J. B., DiAugustine, R. P., and Eling, T. E. (2004) J Biol Chem

279, 6883-6892

ACKNOWLEDGMENTS

We thank Dr. Seung Jun Baek (University of Tennessee, Knoxville, Tennessee) for his helpful discussions. We also thank Dr. Jae-Won Soh (Inha University, Incheon, Korea) and Drs. Kevin Pennington, and Dean Ballard (Vanderbilt University School of Medicine, Nashville, Tennessee) for the kind gift of plasmids. The abbreviations used are: NAG-1, nonsteroidal anti-inflammatory drug-activated gene; TPA, 12-O-tetradecanoylphorbol-13-acetate: PKC, protein kinase C.

FIGURE LEGENDS

Fig. 1 TPA induces NAG-1 mRNA and protein expression in a concentration- and time-dependent

manner in LNCaP cells. (A) TPA induces an increase in NAG-1 protein levels in a concentration-dependent manner. LNCaP cells were treated with indicated concentrations of TPA for 24 hours and total lysates are subjected to western blot analysis for NAG-1. The location of unprocessed (38 kD) and cleaved NAG-1 (16 kD) was shown in arrows. (B) TPA induces an increase in NAG-1 protein levels in a time-dependent manner. LNCaP cells were treated with 10 ng/ml TPA for indicated amount of time and total cell lysates were subjected to western blot analysis for NAG-1. The location of unprocessed (38 kD) and cleaved NAG-1 (16 kD) was shown in arrows. (C) TPA induces an increase in NAG-1 mRNA levels in a concentration-dependent manner. LNCaP cells were treated with indicated concentrations of TPA for 24 hours and total RNA was subjected to northern blot analysis for NAG-1. The membrane was stripped and reprobed for GAPDH to confirm equal loading of RNA. (D) TPA induces an increase in NAG-1 mRNA levels in a time-dependent manner. LNCaP cells were treated with 10 ng/ml TPA for indicated amount of time and total RNA was subjected to northern blot analysis for NAG-1. The membrane was stripped and reprobed for GAPDH to confirm equal loading of RNA. (E) LNCaP cells were transfected with NAG-1 promoter reporter plasmid (-966/70) and pRL-null plasmid. Twenty-four hour after transfection, cells were treated with the indicated concentrations of TPA for 24 hours. NAG-1 promoter reporter activity was normalized to renilla lucifearse activity.

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Fig. 2 TPA induces NAG-1 expression in LNCaP cells in a PKC-dependent manner.

(A) LNCaP cells were pre-treated with 2.5 µM GF109203X, 20 µM PD980509, 20 µM SB203580, or 20 µM SP600125 for 40 minutes and treated with 10 ng/ml TPA for 24 hours. Total cell lysates were subjected to western blot analysis for NAG-1. CTL : no inhibitor (B) LNCaP cells were treated

with 20 µM LY294002 or 10 ng/ml TPA for 24 hours. Total cell lysates were subjected to western blot analysis for NAG-1. The membrane was stripped and reprobed for phospho-Akt and total Akt. CTL : DMSO treated

Fig. 3 TPA induces an increase in NF-κB binding/transcription activity. A) LNCaP cells were treated with 10 ng/ml TPA for the indicated amount of times. Nuclear extracts

were isolated and subjected to gel shift assay using 32P-labeled NF-κB probe. For supershift assay, nuclear extracts at 12 hour after TPA treatment were pre-incubated with anti-p65 antibody. (B)

LNCaP cells were transfected with NF-κB reporter plasmid and pRL-null plasmid. Twenty-four hour after transfection, cells were pre-treated with each inhibitor for 40 min and then treated with 10

ng/ml TPA for 24 hours. NF-κB reporter activity was normalized to renilla lucifearse activity. (C) LNCaP cells were co-transfected with NF-κB reporter plasmid, pRL-null, and empty vector, constitutively active PKC α or constitutively active PKCδ plasmid. NF-κB reporter activity was normalized to renilla lucifearse activity.

Fig. 4 NF-κB p65 binds and activates NAG-1 promoter. (A) LNCaP cells were transfected with NAG-1 promoter reporter plasmid (-966/70), pRL-null plasmid, and expression plasmid for each transcription factor. NAG-1 promoter reporter activity was normalized to Renilla lucifearse activity. (B) LNCaP cells were transfected with p65 expression plasmid, pRL-null, and the indicated NAG-1 promoter reporter plasmid. Forty-eight hour after transfection, cells were harvested and luciferase activity was measured. NAG-1 promoter reporter activity was normalized to renilla lucifearse activity. (C) LNCaP cells were treated with 1 %

formaldehyde in PBS for 10 min. Cells were lysed in lysis buffer and NF-κB p65-associated DNA was immunoprecipitated using NF-κB p65 antibody. NAG-1 promoter region was amplified using PCR with immunoprecipitated DNA or total input DNA as templates. Fig. 5 PKC induces NAG-1 expression in LNCaP cells. LNCaP cells were infected with pRev-Tet-On retrovirus and selected with G418. G418-seleceted pool of LNCaP cells (Tet-On LNCaP) were subsequently infected with pRev-TRE retrovirus encoding each HA-tagged constitutively active PKC isoform and selected with hygromycin/G418. Total cell lysates were subjected to western blot analysis for NAG-1. Membrane was stripped and reprobed for HA and actin.

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Fig. 6 NAG-1 Si RNA expressing LNCaP cells are resistant to TPA-induced apoptosis. (A) Empty vector or NAG-1 Si RNA expressing LNCaP cells were treated with the indicated concentrations of TPA for 24 hours. Total lysates were subjected to western blot analysis for NAG-1. The location of unprocessed (38 kD) and cleaved NAG-1 (16 kD) was shown in arrows. (B) Empty vector or NAG-1 Si RNA expressing LNCaP cells were treated with the indicated concentrations of TPA for 24 hours. Cells were harvested by trypsinization and cell number was counted.

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Minsub Shim and Thomas E. Elingcarcinoma cells

PKC-dependent regulation of NAG-1/PLAB/MIC-1 expression in LNCaP prostate

published online March 9, 2005J. Biol. Chem. 

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